Changes In Extracellular Vesicles Research Articles

Identification of novel blood-based extracellular vesicles biomarker candidates with potential specificity for traumatic brain injury in polytrauma patients.

The goal of this study was to identify changes in extracellular vesicles (EV) surface proteins specific to traumatic brain injury (TBI), which could be used as a diagnostic and prognostic tool in polytrauma patients. Known serum TBI-specific biomarkers (S100B, NSE, and GFAP), which can predict the severity and outcome of isolated TBI, lose their predictive value in the presence of additional extracranial injuries. Extracellular vesicles (EVs) are released from cells in response to various stimuli and carry specific cargo/surface molecules that could be used for tracking injury-responding cells. EVs were isolated using size exclusion chromatography (SEC) from the plasma of two groups of patients (with isolated TBI, ISS≥16, AIShead≥4, n=10; and polytraumatized patients without TBI ISS≥16, AIShead=0, n=10) collected in the emergency room and 48h after trauma. EVs' surface epitope expression was investigated using a neurospecific multiplex flow cytometry assay and compared with healthy controls (n=10). Three enrichments of EV epitopes found to be specific to TBI were validated by western blot. The expression of 10 EV epitopes differed significantly among the patient and control groups, and five of these epitopes (CD13, CD196, MOG, CD133, and MBP) were TBI-specific. The increased expression of CD196, CD13, and MOG-positive EVs was validated by western blot. Our data showed that TBI is characterized by a significant increase of CD13, CD196, MOG, CD133, and MBP-positive EVs in patients' plasma. A high level of MOG-positive EVs negatively correlated with the Glasgow Coma Scale score at admission and could be an indicator of poor neurological status.

Connecting cellular mechanisms and extracellular vesicle cargo in traumatic brain injury.

Traumatic brain injury is followed by a cascade of dynamic and complex events occurring at the cellular level. These events include: diffuse axonal injury, neuronal cell death, blood-brain barrier break down, glial activation and neuroinflammation, edema, ischemia, vascular injury, energy failure, and peripheral immune cell infiltration. The timing of these events post injury has been linked to injury severity and functional outcome. Extracellular vesicles are membrane bound secretory vesicles that contain markers and cargo pertaining to their cell of origin and can cross the blood-brain barrier. These qualities make extracellular vesicles intriguing candidates for a liquid biopsy into the pathophysiologic changes occurring at the cellular level post traumatic brain injury. Herein, we review the most commonly reported cargo changes in extracellular vesicles from clinical traumatic brain injury samples. We then use knowledge from animal and in vitro models to help infer what these changes may indicate regrading cellular responses post traumatic brain injury. Future research should prioritize labeling extracellular vesicles with markers for distinct cell types across a range of timepoints post traumatic brain injury.

Abstract 16961: Characterization of Extracellular Vesicles and RNA Content and Its Relevance to Thrombo-Inflammatory Biomarkers in Patients With Acute Pulmonary Embolism

Introduction: Pulmonary embolism (PE) is a complex disease with significant morbidity and mortality rates globally. Previous studies have shown that extracellular vesicles (EV) carrying disease specific ribonucleic acids (RNA’s) may contribute to pro-inflammatory and pro-coagulant state in VTE. This study aimed on profiling EV related parameters, RNA content, and thrombo-inflammatory markers in patients with acute PE compared to healthy individuals. Materials & Methods: Prospective cohort of patients with PE (n=72) were compared with normal healthy individuals (n=19) and further stratified by PE severity. Blood samples were collected under an IRB-approved project from Loyola University Chicago. Plasma samples were analyzed for the EV parameters, RNA parameters, and thrombo-inflammatory markers (D-Dimer, PAI-1, and CRP) respectively. Results: PE cohort includes 72 patients (41 male, and 31 female) with a median age of 67-years (interquartile range; IQR= 53-76), and a median body mass index (BMI) of 27.5-kg/m 2 (IQR= 24.7-35.7). Compared to controls, EV concentration, and EV/ng RNA were significantly (p= <0.05) lower, while D-Dimer, CRP and PAI-1 were significantly (p= <0.05) higher in patients with PE. In correlation analysis, varying levels of positive and negative correlations between the EV, and RNA parameters, and thrombo-inflammatory markers were noted (Figure 1). Upon stratification on PE severity, D-dimer was higher in massive PE compared to low-risk (p= 0.012), and sub-massive PE (p=0.036) respectively. Conclusion: These studies demonstrate significant changes in extracellular vesicles, RNA content and thrombo-inflammatory markers in acute PE patients compared to healthy controls. These changes may be due to the drivers of VTE pathogenesis or the result of a consumption process in acute PE. Further large scales studies on disease specific exosomes and RNA signatures are warranted to understand their relevance.

Role of plasma extracellular vesicles in prediction of cardiovascular risk and alterations in response to statin therapy in hypertensive patients.

Background:Rapid and accurate new biomarkers to predict risk of cardiovascular disease (CVD) are essential. The utility of extracellular vesicles in predicting the CVD risk is postulated, yet it remains unknown whether their expression is altered in response to statin therapy.Methods:We performed in-vitro studies with human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (hVSMC), and conducted a nested case–control study (nCCS) in hypertensive patients (n = 40) randomized to either atorvastatin or placebo in the ASCOT-LLA. Cases had a major adverse cardiovascular event or death (MACE) during 3.5 years of follow-up (median) from the time of extracellular vesicle characterization while controls, matched for age and duration of treatment, remained event-free. Conditional logistic regression models determined the risk of MACE. Additionally, the relationship of extracellular vesicle levels with statin therapy was assessed.Results:Added to HUVEC, extracellular vesicles increased neutrophil recruitment, and to hVSMC, aggravated calcification and proliferation. In the nCCS, compared with controls, cases (i.e. with MACE) had preceding higher levels of CD14+ and CD14+/CD41+ extracellular vesicles (P = 0.009 and P = 0.012, respectively) and a significant reduction in the median size of the vesicles (P = 0.037). On matched analysis, higher CD14+ extracellular vesicles were associated with a 3.7-fold increased risk of MACE (P = 0.032). Patients treated with atorvastatin (vs. placebo) had both reduced size of extracellular vesicles and the proportion of CD146+ extracellular vesicles (P = 0.034 and P = 0.020, respectively).Conclusion and relevance:These pilot analyses suggest a mechanistic role for extracellular vesicles in the development of CVD, with significant and differential changes in extracellular vesicles amongst those at risk of MACE, and those on atorvastatin therapy.

Optical photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls.

Equine osteoarthritis is a chronic degenerative disease of the articular joint, characterised by cartilage degradation resulting in pain and reduced mobility and thus is a prominent equine welfare concern. Diagnosis is usually at a late stage through clinical examination and radiographic imaging, whilst treatment is symptomatic not curative. Extracellular vesicles are nanoparticles that are involved in intercellular communication. The objective of this study was to investigate the feasibility of Raman and Optical Photothermal Infrared Spectroscopies to detect osteoarthritis using plasma-derived extracellular vesicles, specifically differentiating extracellular vesicles in diseased and healthy controls within the parameters of the techniques used. Plasma samples were derived from thoroughbred racehorses. A total of 14 samples were selected (control; n = 6 and diseased; n = 8). Extracellular vesicles were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy, and human tetraspanin chips. Samples were then analysed using combined Raman and Optical Photothermal Infrared Spectroscopies. Infrared spectra were collected between 950-1800 cm-1. Raman spectra had bands between the wavelengths of 900-1800 cm-1 analysed. Spectral data for both Raman and Optical Photothermal Infrared Spectroscopy were used to generate clustering via principal components analysis and classification models were generated using partial least squared discriminant analysis in order to characterize the techniques' ability to distinguish diseased samples. Optical Photothermal Infrared Spectroscopy could differentiate osteoarthritic extracellular vesicles from healthy with good classification (93.4% correct classification rate) whereas Raman displayed poor classification (correct classification rate = -64.3%). Inspection of the infrared spectra indicated that plasma-derived extracellular vesicles from osteoarthritic horses contained increased signal for proteins, lipids and nucleic acids. For the first time we demonstrated the ability to use optical photothermal infrared spectroscopy combined with Raman spectroscopy to interrogate extracellular vesicles and osteoarthritis-related samples. Optical Photothermal Infrared Spectroscopy was superior to Raman in this study, and could distinguish osteoarthritis samples, suggestive of its potential use diagnostically to identify osteoarthritis in equine patients. This study demonstrates the potential of Raman and Optical Photothermal Infrared Spectroscopy to be used as a future diagnostic tool in clinical practice, with the capacity to detect changes in extracellular vesicles from clinically derived samples.

Contributing to Understand the Crosstalk between Brain and Periphery in Methylmercury Intoxication: Neurotoxicity and Extracellular Vesicles.

Human exposure to methylmercury (MeHg) is currently high in regions such as the Amazon. Understanding the molecular changes associated with MeHg-induced neurotoxicity and the crosstalk with the periphery is essential to support early diagnoses. This work aimed to evaluate cellular and molecular changes associated with behavioral alterations in MeHg acute exposure and the possible changes in extracellular vesicles (EVs) number and S100β content. Adults male Wistar rats were orally treated with 5 mg/kg for four days. Behavioral performance, molecular and histological changes in the cerebellum, and plasma EVs were assessed. MeHg-intoxicated animals performed significantly worse in behavioral tests. MeHg increased the number of GFAP+ cells and GFAP and S100β mRNA expression in the cerebellum but no change in NeuN+ or IBA-1+ cells number was detected. The number of exosomes isolated from plasma were decreased by the metal. S100B mRNA was detected in circulating plasma EVs cargo in MeHg exposure. Though preliminary, our results suggest astrocytic reactivity is displaying a protective role once there was no neuronal death. Interestingly, the reduction in exosomes number could be a new mechanism associated with MeHg-induced neurotoxicity and plasma EVs could represent a source of future biomarkers in MeHg intoxication.

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen.

To identify changes in extracellular vesicles (EVs) secreted by the liver following drug-induced liver injury (DILI), rats were treated with a subtoxic dose (500mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.

Proteomic changes in extracellular vesicles from hepatocytes after cytotoxic ethanol injury

AimsExtracellular vesicles (EVs) play an important role in maintaining normal homeostasis or driving pathogenesis in the liver. For example, various disease‐associated processes (e.g. steatosis, inflammation, fibrosis) are, on the one hand, promoted by EVs produced by hepatocytes after cell stress, injury, or infection yet, on the other hand, inhibited by EVs produced by normal hepatocytes. To better understand these functional differences, proteomic analysis was performed on EVs produced by AML12 mouse hepatocytes maintained in vitro under normal conditions or after exposure to ethanol and TNFα.MethodsAML12 cells were cultured under serum‐free conditions either alone or in the presence of 50mM ethanol for 48 hours plus 30ng/ml TNF‐α over the latter 24 hrs. EVs were purified from conditioned medium by sequential steps of low‐, high‐ and ultra‐ centrifugation. Proteins from two separate EV preparations collected under control (EVNorm) or ethanol/TNFα (EVETOH/TNF) culture conditions were extracted, digested with trypsin, and protein identification was performed using nano‐liquid chromatography‐nanospray tandem mass spectrometry (LC/MS/MS). Sequence data were searched against the Uniprot Mouse database using Mascot Daemon by Matrix Science version 2.6.0 (Boston, MA) Proteins with less than 1% FDR as well as a minimal of 2 significant peptides detected were considered as valid proteins.Results293 proteins were present in duplicate EVNorm samples and 186 proteins were present in duplicate EVETOH/TNF samples. 152 proteins were shared by both EVNorm and EVETOH/TNF and were present at relatively higher levels (quantitative value 10–1000) than the 141 EVNorm‐specific proteins (quantitative value 5–50) or the 34 EVETOH/TNF ‐specific proteins (quantitative values 1–10). Among the most abundant shared proteins, FN, CLUS, ACTG, KPYM and TERA showed diminished levels in EVETOH/TNF versus EVNorm while LG3BP, CFAB, H2AY, RS27A, and IGSF8 showed increased levels in EVETOH/TNF versus EVNorm. GO analysis showed that of the proteins that were unique or enriched in EVNorm or EV ETOH/TNF, some components were shared (exosome, membrane, cytoplasm, nucleus, focal adhesion, extracellular components, cell‐cell adherins, nucleolus, ribonucleoproteins) while other components were unique to either EVNorm (cytoskeleton, mitochondria, myelin sheath, protein complex) or EVETOH/TNF (ribosome, endocytosis, cytosol, Golgi). KEGG analysis showed that the principal pathways for EVNorm‐specific/enriched proteins related to antibiotic synthesis, carbon metabolism, proteasome, actin cytoskeleton, virus infection, focal adhesion, PI3K‐AKT, amino acid metabolism, and glycolysis whereas EVETOH/TNF‐specific/enriched proteins related to ribosome, endocytosis, proteasome, system lupus erythematosus, alcoholism and mineral absorption.ConclusionsEVs from normal or ethanol/TNFα ‐treated hepatocytes show dynamic differences in their respective proteomes. The underlying changes in EV protein composition, both quantitatively and qualitatively, likely contribute to the role that these EV populations play in regulating hepatic homeostasis and the pathogenic outcomes following cytotoxic ethanolic injury in the liver.Support or Funding InformationNIH grants R21 AA025974 and R21 AA023626

Serum extracellular vesicles expressing bone activity markers associate with bone loss after HIV antiretroviral therapy.

We tested whether bone-related extracellular vesicle phenotypes changed after initiating antiretroviral therapy (ART) and determined whether changes in levels of extracellular vesicles correlated with changes in bone mineral density (BMD). Extracellular vesicle phenotypes were measured in blinded serum samples from 15 adults with HIV at baseline, 1, 3, 6 and 12 months after ART initiation. Not all samples were available at each time point so we averaged early (TP1, 1-3 months) and late (TP2, 6-12 months) time points. Extracellular vesicles were stained for osteocalcin (OC), RANKL (CD254), RANK (CD265), M-CSF (macrophage colony stimulating factor), and CD34. Serum OC, procollagen type I N-terminal propeptide (P1NP), and C-terminal telopeptide of type 1 collagen (CTx) were also measured. BMD significantly decreased from baseline to 12 months. Levels of OC+EVs, serum OC, serum P1NP, and CTx were significantly higher at early and late time points compared with baseline. Increases in EVs expressing OC, RANKL, RANK, and CD34 from baseline to TP1 were associated with decreases in total hip BMD from baseline to 12 months. Change in serum OC, P1NP, and CTx from baseline to TP1 or TP2 did not correlate with change in BMD. Early changes in extracellular vesicles expressing markers of bone activity were associated with total hip bone loss 12 months after ART initiation. These data suggest that serum extracellular vesicles may serve as novel biomarkers of bone remodeling. Future studies are required to determine if extracellular vesicles contribute to the effects of ART on changes in bone turnover markers and BMD.